Cloning and expression of Eimeria necatrix microneme5 gene in Escherichia coli

Document Type: Voccinology

Authors

1 Faculty of Veterinary Medicine of Ahvaz

2 Department of Basic Sciences, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran

3 He is working at Veterinary Department of Qum State

4 Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran

5 3Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran

Abstract

Background: Coccidiosis 
caused by Eimeria necatrix has the most economic impact on
poultry production. Micronemal proteins in Eimeria necatrix are thought
to be critical ligands determining host cell specificity at the time of
invasion.               OBJECTIVES: Isolation and purification of Eimeria necatrix oocysts from  Khuzestan province of Iran was performed. A
cDNA encoding microneme 5 (EnMIC5) protein was cloned and expressed as
recombinant protein before the evaluation of its immunogenicity by Western
blotting. METHODS: A primer pair was designed based on the published
nucleotide sequence of Eimeria necatrix LZ strain micronem5 gene. A
Partial cDNA sequence fragment of 758 bp coding for microneme 5 protein
(EnMIC5) was amplified by semi- Nested RT-PCR. PCR products were cloned and
expressed in a Maltose Binding protein (MBP) containing expression vector
(pMAL-c2x) in Escherichia coli. The cDNA which is encoded for 252 amino
acids shows high degree of conservation. It contains the adhesive plasma
pre-kallikrein and seven hydrophilic motifs. RESULTS: The results of SDS-PAGE
revealed that the recombinant protein with a molecular weight of 70 kDa was
over-expressed after induction with IPTG. Western blotting results revealed
that the expressed recombinant protein was reacted with sera of the chicks
infected with Eimeria necatrix. It was suggested that this protein
should have a good immunogenicity and can be used for further studies. CONCLUSIONS: In conclusion, the high degree of sequence homology indicates that this
protein is immunogenic and  might be an
interesting vaccine target, and deserves further investigation

Keywords


Article Title [Persian]

کلونینگ و بیان ژن میکرونم 5 ایمریا نکاتریکس در اشرشیاکلی

Authors [Persian]

  • منصور میاحی 1
  • عباس جلودار 2
  • شهروز مسائلی 3
  • حسین حمیدی نجات 4
  • مسعود صیفی آباد شاپوری 5
  • نغمه موری بختیاری 4
1 دانشکده دامپزشکی دانشگاه شهید چمران اهواز
2 گروه علوم پایه، بخش بیوشیمی و بیوتکنولوژی، دانشکده دامپزشکی دانشگاه شهید چمران اهواز، اهواز، ایران
3 در اداره کل دامپزشکی قم مشغول به مار است.
4 گروه پاتوبیولوژی، بخش انگل شناسی، دانشکده دامپزشکی دانشگاه شهید چمران اهواز، اهواز، ایران
5 گروه پاتوبیولوژی، بخش میکروب شناسی و ایمنی شناسی، دانشکده دامپزشکی دانشگاه شهید چمران اهواز، اهواز، ایران
Abstract [Persian]

زمینه مطالعه:  آیمریا نکاتریکس عامل کوکسیدیوز حداکثر تأثیر اقتصادی را بر روی تولید پرندگان دارد. به نظر می‌رسد پروتئین‌های میکرونم  نقش مهمی در حرکت انگل و تهاجم به سلول‌های میزبان دارد. هدف: جداسازی و خالص‌سازی اووسیست آیمریا نکاتریکس از استان خوزستان-ایران بود. یک cDNA کد کننده پروتئین میکرونم 5 (EnMIC5) کلون و پس از بیان کردن این پروتئین نوترکیب، خاصیت ایمنی‌زایی آن توسط وسترن بلوتینگ مورد آزمایش قرار گرفت. روش کار: یک جفت پرایمر بر اساس توالی نوکلئوتیدی موجود برای ژن میکرونم 5 آیمریا نکاتریکس سویه LZ طراحی و به وسیله آن توالی بخشی از cDNA کد کننده این پروتئین به میزان bp 758 با استفاده از روش Semi- Nested RT-PCR تکثیر گردید سپس قطعه بدست آمده به درون وکتور pMAL-c2x حاوی پروتئین باند شونده به مالتوز کلون و در باکتری E . coli سویه TG1 بیان گردید. قطعه تکثیر شده، یک open reading frame 252 اسیدآمینه ای داشت که به میزان قابل توجهی حاوی مناطق حفاظت شده شامل پری کالکرئین پلاسمایی چسبنده و هفت جزء آبدوست بود. نتایج: در SDS-PAGE پروتئین الحاقی با وزن مولکولی در حدود 70 کیلو دالتون پس از القاء توسط IPTG بیان شد. آزمایش وسترن بلات نشان داد که پروتئین نوترکیب با سرم بدست آمده از جوجه‌های آلوده به آیمریا نکاتریکس واکنش می‌دهد. نتیجه گیری نهایی: یافته‌ها نشان داد پروتئین بدست آمده قدرت ایمنی‌زایی ‌نسبتاً خوبی داشته و می‌تواند در آینده مورد مطالعه بیشتری قرار گیرد.

Keywords [Persian]

  • آیمریا نکاتریکس
  • بیان ژن
  • ایمنیزایی
  • میکرونم ______________________________________________
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